Our long-term objectives are to elucidate fundamental aspects about transcriptional activation in the context of chromatin. A variety of multiprotein coactivators, ATP-dependent (e.g., Swi/Snf) chromatin remodelers and histone acetyltransferases (e.g., SAGA and NuA4), enable the transcriptional apparatus to contend with repressive chromatin structures in eukaryotes. Hence, they are critical to the proper regulation of transcription by RNA polymerase II. This proposal builds on novel molecular approaches that we have developed to increase our understanding of in vivo transcriptional activation mechanisms, using the genetically tractable organism, S. cerevisiae, as a model system. The yeast promoters PH05 and PH08, while coordinately regulated by the same phosphate signaling pathway, have different requirements for transcriptional coactivators. We propose to investigate the molecular basis for these differences by determining how the phenotypic consequences of disruption of coactivator functions varies as a function of promoter occupancy by Pho4. We will also ascertain the extent to which cooperative DNA binding by Pho4 and Pho2, or a functional interaction of their activation domains, leads to additive or synergistic increases in transcription, thereby altering the necessity for individual coactivators at PH05 or PH08. In addition, we will examine whether increased activator binding leads to recruitment of 'excess' chromatin remodelers, establishing functional redundancy, where activation is refractory to inactivation of individual complexes. These studies will utilize chromatin immunoprecipitation and a powerful strategy, TAGM, for quantitative determination of relative activator and coactivator binding in intact cells. We also propose to characterize whether the disruption of multiple nucleosomes at the PH05 and PH08 promoters is sequential, occurring in a random or defined order, or is cooperative. These studies will utilize a novel single-molecule application of our previously published approaches for monitoring chromatin remodeling in vivo with DNA MTases. The studies should lead to important insights into the regulation of transcription. They also have relevance to human health as recent discoveries implicate chromatin and its constituents in cancer and other diseases.